Macular pigment Raman detector for clinical applications

Abstract

Clinical studies of carotenoid macular pigments (MP) have been limited by the lack of noninvasive, objective instruments. We introduce a novel noninvasive optical instrument, an MP Raman detector, for assessment of the carotenoid status of the human retina in vivo. The instrument uses resonant excitation of carotenoid molecules in the visible wavelength range, and quantitatively measures the highly specific Raman signals that originate from the single- and double-bond stretch vibrations of the pi-conjugated carotenoid molecule's carbon backbone. The instrument is a robust, compact device and suitable for routine measurements of MP concentrations in a clinical setting. We characterized and tested the instrument in clinical studies of human subjects to validate its function and to begin to establish its role as a possible screening test for macular pathologies. We also show that the MP Raman spectroscopy technology has potential as a novel, highly specific method for rapid screening of carotenoid antioxidant levels in large populations at risk for vision loss from age-related macular degeneration, the leading cause of blindness of the elderly in the developed world.

Fig. 1

(a) Chemical structure of lutein and zeaxanthin molecules. They differ in the location of the additional C=C bond in one ionone ring. (b) Energy-level diagram of long-chain carotenoid molecules with optical and nonradiative transitions.

Fig. 2

Absorption spectra of excised, flat-mounted human retina (curve a), lutein (curve b), and zeaxanthin (curve c) dissolved in methanol. Note the remarkable similarity of the retinal tissue absorption to the absorption of carotenoid solutions.

Fig. 3

Resonant Raman spectrum of methanol solution of zeaxanthin and lutein measured under excitation with a 488-nm argon laser line. The horizontal dashed line indicates zero for the Raman spectrum of zeaxanthin, which was shifted by 900 counts for the sake of clarity.

Fig. 4

The setup of the fiber-based MP Raman detector, an instrument for quantitative measurements of macular carotenoid pigment content in clinical settings.

Fig. 5

Measuring MP level. The subject looks into the instrument and adjusts the optical alignment while resting the forehead against the device.

Fig. 6

Procedure for alignment of the Raman instrument with the optical axis of the subject’s eye. The subject sees two colored light patterns when looking into the instrument: a weak blue circular disk from the argon laser fiber (shown in gray) and a weak red polka-dot pattern originating from an LED-illuminated light-collection fiber bundle. Alignment is achieved when the subject moves his or her head so that both patterns overlap. (a) Misaligned; (b) aligned.

Fig. 7

Display of a typical Raman measurement of human MP on the computer monitor. See text for details.

Fig. 8

Spectral decomposition of a typical macular spectrum, (curve a) indicating the contributions of retinal autofluorescence (curve b) and a carotenoid Raman spectrum (curve c).

Fig. 9

Measured variation in the Raman signal with pupil diameter for a human subject (circles). The dashed curve is the theoretically expected change in the signal with pupil diameter. Above a 7-mm pupil diameter, the signal stays constant (see text).

Fig. 10

Calibration curve of the MP Raman detector linking the instrument reading with the carotenoid content in a human macula. The curve was obtained using a model eye discussed in the text containing zeaxanthin dissolved in methanol. The sampling volume, 1 μl, corresponds to a 1-mm-diameter spot at a macula and contains carotenoid typical for a human macula.

Fig. 11

Repeatability of Raman measurements. The left part represents data for zeaxanthin solution in a model eye. The central and right data represent the short-term and long-term repeatability of macular pigment measurements for two different subjects. The Raman intensities for the long-term dataset were measured over a 2-week period in five sessions.

Fig. 12

Relative variation in the laser light intensity at the retina with the optical power of correcting spectacles or contact lenses. These curves should be used on a regular basis for adequate correction of MP Raman data when the subject has used his or her lenses during MP measurements.